Fluorimetric determination of hydrogen peroxide production by the haemocytes of the wax moth Galleria mellonella (Lepidoptera: Pyralidae)

The aim of this study was to investigate whether haemocytes of Galleria mellonella (Lepidoptera: Pyralidae) larvae produce reactive oxygen species (ROS) like human blood phagocytes.The production of ROS was measured first using luminolenhanced chemiluminescence of un-stimulated and stimulated (four activators with different modes of action) haemolymph or isolated haemocytes.However, spontaneous and activated production of ROS remained at the background level.In subsequent experiments an ultrasensitive fluorescence method using Amplex Red reagent to detect hydrogen peroxide (H2O2) was used.After optimization, Amplex Red was successfully used for determining H2O2 production by both un-stimulated and stimulated haemocytes.To determine the affect of pH and ions on the measurement, several diluent solutions were tested.This revealed that Ca 2+ and Mg 2+ ions are less important for the reaction in insect than mammalian cells.Among the activators tested, phorbol myristate acetate (PMA) and calcium ionophore (Ca-I) had the best stimulatory effect on insect samples, while opsonised zymosan particles (OZP) was the best activator for human phagocytes.In conclusion, the haemocytes of G. mellonella produce H2O2 as an important innate immunity factor, but under different conditions and in different amounts, which probably results in them being less effective in killing microbes than human phagocytes.